Expression and identification of recombinant human heat shock protein70 in pichia pastoris

Objective To efficiently express human heat shock protein70 (rHSP70) in pichia pastoris, and to prepare recombinant hHSP70 (rhHSP70) with the same structure and activity as natural hHSP70.Methods hHSP70 DNA was cloned from human genomic DNA by PCR to construct eukaryon expression vector pPICZα/hHSP70. The recombinant vector was transformed into the pichia pastoris via electroporation after sequencing. The transforming positive clones were screened by PCR, and rhHSP70 in culture supernatant induced by methanol was identified by Western blot to screen the engineer bacteria with high expressions.Results The sequence of hHSP70 DNA obtained was identical with that of cDNA published on GeneBank. SDS-PAGE and Western blot analysis showed there were rhHSP70 in the culture supernatant induced by methanol with 72 kD of molecular weight. Sequence analysis of amino acid approved that 15 amino acids at amido-rhHSP70 were identical with that of natural rhHSP70, and expression levels of rhHSP70 reached at 250 mg/L.Conclusions Pichia pastoris engineer bacteria with high effective expression of rhHSP70 could be prepared.